Myxoma virus mutants for cancer treatment

ABSTRACT

Myxoma viruses that are deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M 136, M-T4 and M-T7 are useful for treating cancer.

BACKGROUND OF THE INVENTION

The use of certain genetically modified myxoma viruses for treating cancer is disclosed in WO 04/078206 (Robarts Research Institute).

SUMMARY OF THE INVENTION

This invention relates to Myxoma viruses (MV) that are deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M136, M-T4 and M-T7. Such viruses are used in a method for and in the manufacture of a medicament for, inhibiting a cancer cell, which method comprises administering to the cell an effective amount of the Myxoma virus. They are also used in a method for and in the manufacture of a medicament for, treating a human subject having cancer, comprising administering to the patient an effective amount of the Myxoma virus. This invention also provides a pharmaceutical composition comprising such Myxoma viruses and a pharmaceutically acceptable carrier, as well as a kit comprising such Myxoma viruses and instructions for treating a cancer patient.

DESCRIPTION OF THE FIGURES

FIG. 1. Endogenous activated Akt levels in human glioma cells

FIG. 2. Viral replication efficiency of the various vMyx-hrKOs and controls in human glioma cell lines.

FIG. 3. Secreted early and late viral gene expression indicates that some of the vMyx-hrKO are unable to transit from early to late gene expression.

FIG. 4. Selected single step growth curves.

FIG. 5. Cell-based cytotoxicity assay

DETAILED DESCRIPTION OF THE INVENTION

WO 04/078206 (Robarts Research Institute) discloses the use of certain genetically modified myxoma viruses for treating cancer. This invention represents an advance by providing more specific modified myxoma viruses for such uses. The techniques disclosed therein are applicable generally to the myxoma viruses of this invention and the contents of WO 04/078206 are incorporated herein by reference.

As used herein “deficient in the activity of” a given Myxoma virus protein means that the virus has less of the activity in question than wild-type Myxoma virus. “Substantially no activity” of a given viral protein means that the virus has no detectable level of such activity. Examples of Myxoma viruses having substantially no activity of a given viral protein include mutants in which the gene for such protein has been deleted or otherwise knocked-out.

In accordance with this invention, any kind of cancer or cancer cell can be inhibited or treated. In an embodiment of this invention, the cancer cell is a mammalian cancer cell. In a more specific embodiment, the cancer cell is a human cancer cell. Examples of such human cancer cells include gliomas.

It has been demonstrated that wild-type myxoma virus (vMyxgfp) can produce a productive, long-lived infection, and destroy and clear implanted tumor tissue when injected intratumorally into human gliomas implanted in murine brains (Lun et al, 2005 Cancer Research 65:9982-9990). As well, a screen of the NCI-60 reference collection indicated that MV productively infects the majority (15/21) of human tumor cells tested (Sypula et al. 2004 Gene Ther. Mol. Biol. 8:103-114). To expand understanding of MV tropism in cancer cells, a series of human glioma cells (U87, U118, U251, U343, U73) that were previously tested for wild-type MV permissiveness were screened. These findings have been extended in the following Examples by testing the infection and replication of several MV viruses in which specific host range genes, identified as having a role in defining MV tropism in rabbit cells, have been deleted. These viruses are collectively referred to as host range knockouts (vMyx-hrKO). Variation was observed in the ability of various vMyx-hrKOs to replicate and spread in the human glioma cells. vMyxT2 (U251), vMyxT4KO (U87, U118, U251 and U373) and vMyxT5KO (U251, U373) exhibited some restriction in specific human gliomas. In contrast vMyx63KO and vMyx135KO appeared to replicate and kill more effectively in several of the gliomas.

The invention will be better understood by reference to the following examples, which illustrate but do not limit the invention described herein.

EXAMPLES Example 1

Fifty micrograms of whole cell lysates were probed with antibodies against phosphorylated Akt at positions threonine 308 (P-Akt T308) and serine 473 (P-Akt S473) or total Akt. Based on the levels of activated Akt U87 and U343 would be expected to be infectable and U118, U251 and U373 to be more resistant to MV infection. (FIG. 1).

Example 2

Virus stocks were titrated on the various glioma cells and control BGMK or RK13 cells. Virus titres, derived from the gliomas, were compared to the control levels and a value of viral replication efficiency was estimated. Based on these results none of the gliomas supported viral growth to the levels observed in the control lines. However some viruses (vMyx135KO, vMyx63KO and vMyx136KO) were more efficient than other knockouts. As well, some glioma lines supported more replication (U87, U343 and U373). (FIG. 2)

Example 3

Various human gliomas were infected with a range of vMyx-hrKOs. Twenty hpi the infected supernatants were collected and concentrated 10×. Fifteen microlitres of concentrated sups were separated on a 12% SDS-PAGE gel and probed with anti-Serp1 (mAB; late MV gene product). The blots were stripped and probed for early gene expression with anti-M-T7 (pAB; early MV gene product). The results suggest that several vMyx-hrKOs are restricted in their transit from early to late gene expression including vMyxT2KO, vMyxT4KO and vMyxT5KO. And in two glioma lines (U87 and U37), vMyxT4KO does not even undergo early gene expression. (FIG. 3).

Example 4

Cells were seeded in 48 well dishes and infected cells were collected at the times indicated. Virus was released from the collected cell pellets and titrated back onto BGMK cells. Although there was replication of the tested viruses, the best amplification appeared to occur in the U87 and U343 cells. (FIG. 4).

Example 5

The ability of the various vMyx-hrKOs and control viruses to have a killing effect in the panel of human gliomas was tested by a cytotoxicity assay. The appropriate cells were seeded in 96 well dishes and 24 h later were infected with the viruses at various MOIs. Seventy-two hours post infection the infected cells were treated with the WTS reagent (Roche) to measure cell viability. Colour changes were measured at 450 nm every 60 minutes for 4 hours. Uninfected control wells were used to determine normal proliferation and a blank well served as a background control. (FIG. 5). 

1. A method for inhibiting a cancer cell comprising administering to the cell an effective amount of a Myxoma virus that is deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M136, M-T4 and M-T7.
 2. The method of claim 1, wherein the Myxoma virus has substantially no activity of the Myxoma virus protein.
 3. The method of claim 1, wherein the cell is a mammalian cancer cell.
 4. The method of claim 3, wherein the cell is a human cancer cell.
 5. The method of claim 4, wherein the cell is a glioma cell.
 6. A method for treating a human subject having cancer, comprising administering to the patient an effective amount of a Myxoma virus that is deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M136, M-T4 and M-T7.
 7. The method of claim 6, wherein the Myxoma virus has substantially no activity of the Myxoma virus protein.
 8. The method of claim 6, wherein the cancer is a glioma. 9-18. (canceled)
 19. A pharmaceutical composition comprising a Myxoma virus and a pharmaceutically acceptable carrier, wherein the Myxoma virus is deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M136, M-T4 and M-T7.
 20. The pharmaceutical composition of claim 19, wherein the Myxoma virus has substantially no activity of the Myxoma virus protein.
 21. The pharmaceutical composition of claim 19, wherein the cancer is a glioma.
 22. A kit comprising a Myxoma virus and instructions for treating a patient having cancer, wherein the Myxoma virus is deficient in the activity of a Myxoma virus protein selected from the group consisting of M11L, M063, M136, M-T4 and M-T7.
 23. The kit of claim 22, wherein the Myxoma virus has substantially no activity of the Myxoma virus protein.
 24. An isolated Myxoma virus that is deficient in the activity of a Myxoma virus protein selected from the group consisting of M063 and M136.
 25. The isolated Myxoma virus of claim 24, wherein the Myxoma virus has substantially no activity of the Myxoma virus protein. 